The recent rapid advances in genetic engineering triggered the development of various techniques in molecular biology. This has been commensurate with remarkable advances in the analysis of genetic information and the unravelling of functions of genes and many attempts are being made to exploit these achievements in practical therapeutic settings. One of the areas that have seen the most remarkable advances is that of gene therapy. Etiological genes of various genetic diseases have been discovered and deciphered on one hand, and procedures for transferring such genes into cells by physical and chemical techniques have been developed on the other; as a result, gene therapy has progressed from the stage of preclinical experimentation to practical clinical applications.
Depending on the type of cells (target cells) for gene transfer, gene therapy is classified as either germline cell gene therapy or somatic cell gene therapy. Another way of classification is into augmentation gene therapy which involves the addition of a new (normal) gene, with an abnormal (etiological) gene left intact, and replacement gene therapy for replacing an abnormal gene by a normal gene. At the present stage, only augmentation gene therapy of somatic cells is being practised in consideration of ethical and technological restraints. More specifically, a method of gene therapy in current practice is one by autotransplantation (ex vivo gene therapy) in which a target cell is taken out of the patient and a gene to be inserted is transferred into the target cell, which is then replaced into the patient's body. A method under review for future possibility is one that involves direct gene administration into the patient (in vivo gene therapy).
One of the major technological challenges for the clinical application of the above-described gene therapy is the development of a method for introducing an exogenous gene into a target cell in an efficient and consistent way. In the early eighties, physical techniques such as microinjection were attempted; however, these methods were not eventually commercialized for several reasons such as low gene transfer efficiency, incapability to achieve consistent transfer and the limitations of the then available technology of large-scale cell cultivation. It was not until a recombinant virus (viral vector) for inserting an exogenous gene efficiently into a target cell was later developed that the clinical application of gene therapy became a possibility.
In the United States of America, about 70 protocols of gene therapy have been approved and in practice no fewer than 200 patients are presently undergoing gene therapy. A mouse leukemia virus (MoMLV, or Moloney's murine leukemia virus) vector is the most commonly used means of gene transfer. MoMLV is a kind of retrovirus and infects a host cell if the envelope on its surface binds specifically to the receptor on the surface of the host cell. Recombinant MoMLVs are capable of gene transfer into different cell species depending on the type of envelope and are classified as, for example, ecotropics which infect only rodents and amphotropics which infect both rodents and human cells.
Preparing a recombinant MoMLV vector first requires the construction of two plasmids, one being a helper plasmid which comprises gag, pol and env genes to be coded in a MoMLV genome and a promoter for driving these genes, and the other being a vector plasmid having the terminal repeated sequence (LTR) of MoMLV inserted at both ends of the gene acting as a drug. In this case, for the purpose of preventing the production of a wild type of virus, a packaging signal which is a signal sequence for packaging the gene into the particle of a virus is preliminarily removed from the helper plasmid. In contrast, the packaging signal is contained in the vector plasmid. In many cases, a reporter gene for recognizing or selecting only the cell transfected with the viral vector is inserted into the gene sequence of the vector plasmid. Cotransfecting the cell with these two kinds of gene allows a recombinant viral vector to be produced within the supernatant of culture.
In recent years, cell lines called "packaging cells" have been established as means for achieving more efficient preparation of MoMLV vector. These are cell lines in which the helper plasmid and/or vector plasmid which are necessary for preparing the MoMLV vector have been integrated stably into the cellular genomic DNA. The use of such cell lines offers many advantages, among which the following are worth particular mention:
(1) the cumbersomeness of transfection in the preparation of a viral vector is eliminated; PA0 (2) compared to transfection procedures typified by a calcium phosphate procedure which are limited in the efficiency of gene transfer into a cell, using packaging cells into all of which a gene of interest has already been transferred Is advantageous for preparing a viral vector of high potency; PA0 (3) if transfection is performed in several lots by the calcium phosphate procedure, the gene transfer efficiency scatters between lots, so viral vectors of a constant potency cannot be supplied consistently but this is not the case with the preparation procedure using packaging cells and the scattering of gene transfer efficiency is small; and PA0 (4) in the calcium phosphate procedure, preparing a large quantity of viral vectors requires a correspondingly large volume of plasmids for transfection but there is no need for this if the packaging cells are used.
Because of these and other advantages, the use of the packaging cells is presently the most common method of preparing MoMLV vector.
Another property of MoMLV vector is the lack of host specificity. This property indicates that various cells can be the target cell of MoMLV vector, making it possible to use the vector in association with various diseases. On the other hand, the inherent lack of host specificity which is the property of the viral vectors under consideration makes their administration to the human body difficult,
As a matter of fact, if these viral vectors are to be utilized for therapeutic purposes, certain considerations must be given to the method of administration. For instance, when they are to be administered to hemocytes, the method in current use comprises the steps of taking the cell to be treated out of the human body, performing gene transfer and thereafter putting the cell back into the human body (ex vivo gene transfer); however, this technique requires special equipment and hence can be employed for therapeutic purposes in only limited facilities. If the viral vectors are to be administered to cells that have settled in certain organs or tissues, the method currently used is topical administration to the organ or tissue to be treated; however, this approach has several problems such as the need to perform a surgical operation for enabling the topical administration.
Tissue specific viral vectors have been developed as a means for solving the aforementioned problems. An HIV vector is a kind of tissue specific viral vectors capable of specific gene transfer into CD4 positive cells and utilizes the ability of HIV envelope protein gp120 to bind specifically to the CD4 protein on the surfaces of CD4 positive T lymphocytes during infection (Shimada, T., et al, J.Clin. Invest. 88, 1043, 1991). Diseases that are anticipated to be treated by gene therapy using this viral vector include acquired immunodeficiency syndrome (AIDS) and adult T-cell leukemia (ATL) which have particularly high mortality rates because CD4 positive lymphocytes are the etiological agent and for which there are no established methods of treatment.
Acquired immunodeficiency syndrome (AIDS) is a disease caused by infection of CD4 positive lymphocyes with human immunodeficiency virus (HIV) and, as the result of severe damage to cell-mediated immunity, it triggers the onset of various opportunistic infections, lymphoma, neuropathy, etc. The presently used therapeutics of HIV are drugs categorized as reverse transcriptase inhibitors of a nucleotide class and 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), etc. are used either independent-ly or in combination. However, none of these drugs have an ability to reject already infected cells; in addition, they have big problems such as the one of causing severe side effects including significant bone marrow suppression and symptoms in digestive organs, as well as the occurrence of drug-resistant viral strains; under the circumstances, there is a strong need to develop a new type of drug that is more efficacious, has less side effects and permits fewer resistant viral strains to occur.
Speaking of adult T-cell leukemia (ATL), this is a conceptual disease proposed by Uchiyama et al. (Uchiyama, T. et al., Blood, 50, 481-492, 1977) and HTLV-1 is considered to be the etiological virus of the disease (Hinuma, Y. et al., Proc. Natl. Acad, Sci. USA, 78, 6476-6480, 1981); however, much remains to be elucidated as regards the mechanism of the onset of ATL and the mechanism of growth of ATL cells. As far as acute lymphocytic leukemia is concerned, combinations of chemotherapy with radiotherapy and/or bone marrow transplantation have proven today to cause a fairly high incidence of remission or healing. However, as for ATL, the treatment by those methods has not achieved satisfactory results and the development of a new therapeutic method is in urgent demand.
In recent years, the applicability of gene therapy to these refractory diseases has been under preclinical review. As for AIDS, various treatment systems that take advantage of the mechanism of HIV replication in elegant ways have been proposed and they include: using RNA decoys such as TAR and RRE decoys in order to suppress the actions of Tat and Rev which are believed to be potent accelerators of HIV replication (Sullenger, B. A. et al., Cell 63, 601, 1990); hybridizing with HIV mRNA or DNA by means of an antisense oligonucleotide (Chatterjee, S. et al., Science, 258, 1485, 1992); cutting the RNA of HIV by means of a ribozyme (Stevelo, K. M. et al., Virology, 190, 176, 1992);and using a transdominant mutant to suppress the function of proteins essential to HIV replication (Hope, T. J. et al., J. Virol, 66, 1849, 1992).
As for ATL, a system is under review that integrates a thymidine kinase gene in a human simplex herpes virus into an ATL cell, said gene being expressed such that only the ATL cell is specifically killed by means of ganciclovir (Kazunori Imada and Taku Uchiyama, ZOKETSU INSHI (Hematopoietic Factors), 5, 4, 501-503, 1994).
As already mentioned, the HIV vector has very high utility as a tissue specific viral vector but it has several problems with the procedure of preparation. The currently employed method of preparing HIV vector is by cotransfecting COS cells with a helper plasmid and a vector plasmid just prior to use. However, as noted hereinabove, the transfection procedures typified by a calcium phosphate method has various problems including (1) cumbersome operations, (2) limited efficiency of gene transfer, (3) lot-to-lot fluctuations in the efficiency of gene transfer and (4) the need for large-scale preparation of plasmids. Hence, it has been necessary to establish a method capable of more efficient and consistent preparation of the HIV vector.
Although the HIV vector holds promise for use as a tissue specific viral vector, the method of its preparation involves many problems and the currently employed method is by cotransfection with a helper plasmid and a vector plasmid just prior to use. However, several big problems exist with this method, as summarized below: (1) transfection efficiency is variable from lot to lot, so the potency of the vector fluctuates to make consistent supply impossible; (2) due to the cumbersomeness of transfecting operations, vectors cannot be prepared in large quantities; and (3) if large amounts of HIV vector are to be prepared, correspondingly large amounts of plasmid vector have to be prepared. As for MoMLV vector, packaging cell lines have already been established that have a helper plasmid and a vector plasmid incorporated stably into genomic DNA; however, the establishment of packaging cell lines has not yet been accomplished for HIV vector. A reason for this would be that compared to cells for sustaining MoMLV which are mouse-derived such as murine 3T3 cells, human-derived cells have to be used as cells for sustaining HIV vector.